Methods of cell breakage

It was imperative to find a method w h i c h gave maximal cell breakage, such that future cell wall isolation from low biomass yield iron limited cells would not b e impeded. Two alternative cell breakage methods w ere investigated, these were:- a) flash freezing, b) Braun homogenization.

Unfortunately flash freezing did not c a u s e adequate cell breakage. Three cycles of freezing in l i q u i d nitrogen and thawing at 55 *C was incapable of lysing m o r e than 1% of the total cells visualized under microscopic examination.

Braun homogenization proved more ho p e f u l when upto 0.2 g wet weight of cells per tube were homogenised using 0.1-0.11 nm glass beads over a 15 min period with c o n s t a n t cooling. Microscopic examination after this time p e r i o d showed that a large proportion of the cells had been disrupted. Any unbroken cells were usually re-homogenised in order to obtain maximum cell disruption.

4.2.4 Cell ««all isolation by differential centrifugation (Woitzik et a l . , 1988) 4.2.4.1 Method 4

Synechococcus W H 7803 cells were broken by Braun homogenization in 20 m M Tris-HCl pH 8.0 (section 4.2.3.3). The homogenate was washed from the beads and centrifuged at 3.000 rpm for 40 min in a Gallenkamp bench centrifuge to remove unbroken cell debris. The supernatant was re­ centrifuged initially at 3,000 rpm in a Gallenkamp bench centrifuge, for 5 min, to remove any contaminating whole cells, then at 18,000 rpm, for 30 min in a MSE HiSpin centrifuge using an 8x10 fixed angle rotor. The deep purple supernatant contained the cytoplasmic fraction, the purple colouration was due t o the phycobilisomes. The supernatant was decanted and the pellet containing the cell envelopes was washed twice in 20 m M Tris-HCl pH 8.0, by centrifugation at 18.000 rpm in an 8x10 fixed angle rotor in a MSE HiSpin centrifuge for 30 min. The cell envelopes were resuspended initially in the residual volume of 20 mM Tris-HCl (pH 8.0) buffer then loaded (5 ml volume) onto a discontinuous sucrose gradient [4 ml 55, 50, 40% [w/v] sucrose and 2 ml 30% [w/v] sucrose in 20 mM Tris-HCl buffer p H 8.0]. The tubes were centrifuged at 16,900 rpm for 12 hr at 4*C using a swing out bucket Beckman SW28 rotor, in a Beckman L8 ultracentrifuge. Cell walls w ere isolated from the 55% [w/v] sucrose band by resuspension in 20 m M Tris-HCl (pH 8.0) buffer followed b y centrifugation in a M S E centrifuge at 33,000 rpa in a 10x10

rotor for 30 min at 4*C. The cell wall pellet was purified further by discontinuous sucrose density centrifugation, and the putative cell wall fraction recovered from the 55% [w/v] sucrose fraction as described previously. The putative cell wall pellet was resuspended in 5 ml 20 mM Tris-HCl (pH 8.0) buffer containing 10 m M M gCl2 . An equal volume of 4% [v/v] Triton-X-100 in 20 mM Tris-HCl (pH 8.0) buffer was added and the Triton-soluble components extracted from the cell walls by stirring for 20 min at 2 3 *C. Triton-insoluble cell walls were isolated by centrifugation at 33,000 rpm for 60 min in a 10x10 rotor in a MSE centrifuge. The resulting supernatant was olive green in colour, similar to the Triton-soluble supernatant described in section 4.2.2 and the cell wall pellet was a similar rust-red colour. The putative cell wall pellet was washed in extraction buffer [20 mM Tris-HCl (pH 8.0), 10 mM MgCl2 ] excluding Triton-X-100 and resuspended in 500 Ml 20 mM Tris-HCl buffer pH 8.0.

4.2.4.2 Results

A rust-red cell wall pellet was obtained by this alternate method similar to that described in section 4.2.2. This suggests that the 70%-80% [w/v] sucrose band obtained previously were also cell walls. Cell wall colour can vary between orange, red or yellow once devoid of thylakoid and cytoplasmic membranes (Heckesser & Jurgens, 1988). SDS-PA6E analysis and Coomassie staining (Figure 4.2.5) of samples taken throughout the isolation procedure showed the separation of the cytoplasmic and membrane fractions by

centrifugation after cell disruption (track 1 and 2). However, a lot of the cell wall protein present in the cell envelope fraction (track 2) was lost during discontinuous sucrose density centrifugation. This was indicated by the decrease in the major polypeptide bands in the 55% [w/v] sucrose gradient (track 4). The majority of t h e cell wall material had been pelleted, this is highlighted by the sample in track 3. Track 3 contains protein taken from the pellet obtained after the initial sucrose density purification step, and subsequently treated in exactly the same w a y as the putative cell wall fraction in track 5.

Comparison of cell wall fractions obtained by this method (track 5 and 3) compared with method 3 (track 6) indicates that method 3 is able t o give a greater cell wall yield (track 6 vs track 5) and that t h e cell wall fraction contains less contaminating protein (t rack 6 vs track 3).

Apart from the fact that the cell breakage technique and the buffers employed in method 4 were more favourable, the more efficient cell wall isolation procedure of method 3 was preferred. However, a compromise was eventually decided upon which incorporated aspects of both methods 3 a n d 4. This method (see Materials a n d Methods) was eventually used to isolate internal membranes and cell walls from iron-depleted cultures of Synechococcus WH 7803 (Chapter 6).

Figure 4.2.5 Synechococcus WH 7803 outer aeibranes isolated by differential centrifugation and detergent extraction (Method 4)

6-20% [w/v] exponential gradient SDS-PAGE after silver staining. Tracks: 1) Cytoplasmic fraction. 2) Cell envelope fraction prior to discontinuous sucrose density centrifugation 55-30% [w/v] sucrose. 3) Cleaned cell wall fraction obtained from the pellet after the initial discontinuous sucrose density centrifugation, and treated as described for the putative cell wall fraction recovered from the 55% [w/v] sucrose band. 4) Cell wall sample fro m the 55% [w/v] sucrose band after t h e initial sucrose density centrifugation procedure. 5) Putative cell wall fraction obtained using method 4. 6) Putative cell wall fraction obtained using method 3. M ,.x1 0 "3 1 2 3 4 5 6 94 Ü 1» - 67 m ■ ;

I

1

4 . 3 C e l l w a l l c h a r a c t e r i z a t i o n

4.3.1 Use of scanning spectroscopy to identify the cell wall fraction

The absorption spectrum of the isolated cell wall fraction (Figure 4.3.1a) showed absorption maxima at 392 nm, 430 nm, 490 nm, which is indicative of carotenoids. However, there was little or no contamination with chlorophyll a (670 nm) or phycobiliproteins (545 nm, 620 nm, 650 nm) in these cell wall preparations. The presence of carotenoids in the cell walls of cyanobacteria is now accepted (Jurgens & Weckesser, 1985). Extraction of the inner membrane fractions with Triton-X-100 failed to remove the absorption maxima indicative of carotenoids, suggesting that carotenoids were a true component of the cell wall and not due to contamination by the cytoplasmic membrane. The absorption spectrum of fractions taken from the top of the sucrose gradient (Figure 4.3.1b) showed absorption m a xima at 438 nm, 497 nm possibly due to cytochromes and carotenoids associated with the thylakoid and cytoplasmic membranes, and also at 545 nm, 628 n m - due to phycobili proteins and 670 nm - due to chlorophyll a, both of which are associated with the thylakoid membrane. Carotenoids extracted from the cell wall can also be identified by their Rf values b y thin-layer chromatography (Weckesser & Jurgens, 1988).

a) Isolated Synschococcus WH 7803 cell wall fraction banding at the 80-70% [w/v] sucrose interface, b) Fraction front the top of the gradient, above the 50% [w/v] sucrose layer.

F i g u r e 4 . 3 . 1 A b s o r p t i o n s p e c t r u n o f f r a c t i o n s f r o s t h e s u c r o s e g r a d i e n t 5 0 - 8 5 % [w/v]